Journal
ACS SYNTHETIC BIOLOGY
Volume 4, Issue 4, Pages 383-392Publisher
AMER CHEMICAL SOC
DOI: 10.1021/sb500247a
Keywords
hydroxyproline; hydroxy-L-pipecolic acid; hydroxylation; proline hydroxylase; protein engineering
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Funding
- Academic Sinica
- National Synchrotron Radiation Research Center (Taiwan, ROC)
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Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Pro line hydroxylases are representative members of the nonheme Fe2+/alpha-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of L-proline into hydroxy-L-prolines (Hyps). L-Proline cis-4-hydroxylases (cis-P4Hs) from Sinorhizobium rneliloti and Mesorhizobium loti catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-Hypip and cis-3-Hypip. To selectively produce cis-5-Hypip without simultaneous production of two isomers, protein engineering of cis-P4Hs is required. We therefore carried out protein engineering of cis-P4H to facilitate the conversion of the majority of L-Pip into the cis-5-Hypip isomer. We first solved the X-ray crystal structure of cis-P4H in complex with each of L-Pro and L-Pip. Then, we conducted three rounds of directed evolution and successfully created a cis-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward cis-5-Hypip.
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